Resistance to physiological disorders in lettuce

ABSTRACT

Provided is a method for screening a population of plants for the presence therein of individuals that show a reduced susceptibility to ethylene and physiological disorders, in particular Russet Spotting and Yellowing, as compared to a control plant, wherein a population of seeds is germinated in darkness and in the presence of ethylene to obtain seedlings that, when having a longer hypocotyl as compared to the original ethylene-sensitive control under ethylene, are selected as plants showing a reduced susceptibility to ethylene and physiological disorders, in particular Russet Spotting or Yellowing. Also provided are plants thus selected.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/829,047 filed Jul. 1, 2010 which is a divisional of U.S. applicationSer. No. 11/650,834 filed Jan. 8, 2007, which claims priority to EPapplication no. 2006075040, filed Jan. 6, 2006. The foregoingapplication, all documents cited in the foregoing application(“application documents”) and all documents cited or referenced in theapplication documents are incorporated herein by reference. Also, alldocuments cited in this application (“herein-cited documents”) and alldocuments cited or referenced in herein-cited documents are incorporatedherein by reference. In addition, any manufacturer's instructions orcatalogues for any products cited or mentioned in each of theapplication documents or herein-cited documents are incorporated byreference. Documents incorporated by reference into this text or anyteachings therein can be used in the practice of this invention.Documents incorporated by reference into this text are not admitted tobe prior art.

FIELD OF THE INVENTION

The invention relates to a method for screening a population of plantsfor individuals that are altered with respect to their mode of ethyleneresponse. The invention further relates to plants and plant parts, inparticular leafy vegetables, thus identified. More in particular, thisinvention relates to lettuce (Lactuca sativa L.) that shows an alteredresponse to ethylene which leads to a reduced susceptibility of thiscrop species to physiological disorders such as Russet Spotting andYellowing. The invention also relates to seeds and progeny of theseplants and plant parts.

BACKGROUND OF THE INVENTION

Breeding of leafy vegetables like lettuce aims at the production ofcommercial varieties optimally adapted to produce marketable products.Many characteristics need to be taken into account during selectionwhich relate to both input and output traits. One of the most importanttraits in this respect relates to post-harvest quality, in particular toshelf life. The avoidance of physiological disorders and more inparticular Russet Spotting and Yellowing are important elements that canextend the shelf life of a lettuce crop or parts thereof.

Ethylene is a plant hormone generally known to stimulate physiologicalprocesses related to senescence. In lettuce this stimulation becomesapparent through the formation of symptoms such as Russet Spotting andYellowing.

The Russet Spotting disorder is characterised by the appearance of brownspots alongside the midrib of the leaves whereas Yellowing is thegeneral bleaching of leaves which occurs during senescence as aconsequence of chlorophyll breakdown.

Although mature heads of lettuce are known to produce only minuteamounts of ethylene, the plants are highly sensitive towards this planthormone. Therefore, physiological disorders associated with ethylenesensitivity which reduce the post-harvest quality of lettuce are mainlycaused by external sources of ethylene. Exposure to such externalsources can occur during harvesting, processing and storage of theproduce.

For example, when lettuce is transported or stored in the vicinity ofethylene producing fruits such as apples, pears or peaches severedeterioration may occur. Furthermore, when lettuce is processed and usedin packaged fresh-cut mixtures there may be limitations with respect tothe ingredients which can be used due to ethylene release by one or moreof the ingredients.

Russet Spotting is a physiological disorder which is manifested by theappearance of numerous brown spots along the midrib of the leaf. Thebrowning symptoms can spread all over the leaf during the progressivestages of the disorder. Russet Spotting is known to occur especiallywhen mature lettuce heads are stored at lower temperatures (5° C.) inthe presence of low concentrations (ppm levels) of ethylene.

Symptom formation can be antagonised by applying the plant hormone auxinor calcium. Furthermore, modified atmospheres containing low oxygenlevels reduce the speed at which symptoms develop.

At the biochemical level Russet Spotting appears to develop as aconsequence of a local stimulation of lignin biosynthesis, which causeslignification and cell wall thickening around the area of the leaf wherethe visual symptoms will appear.

The brown discolouration is caused by the stimulation of phenolicmetabolism. The enzyme phenylalanine ammonia lyase (PAL), which has beenshown to be induced by ethylene, catalyses the first committed step ofthe phenylpropanoid pathway. Phenolic compounds which are formed mainlyinclude caffeic acid derivatives as well as a number of flavonoids suchas (+)catechin and (−)epicatechin. Subsequent oxidation of thesecompounds by polyphenol oxidase (PPO) leads to the brown discolourationtypically observed in Russet Spotting. Finally, the symptoms may becomemore severe due to collapsing of tissue and cell death.

Senescence is a naturally occurring, developmental process at the end ofa life cycle of a plant or plant organ during which metabolism isreprogrammed in order to remobilize resources into reproductivestructures like seeds. Although senescence is a developmental processcaused by endogenous factors like physiological age, there are manyexogenous factors which can modulate senescence.

Yellowing of leaves, the most visible symptom of senescence, is aconsequence of chlorophyll breakdown during a relatively late stage ofsenescence, which can be enhanced by ethylene once a leaf is receptive.Well-known other stimulating factors of senescence are wounding,darkness and nutrient deficiency. Although ethylene is the mostimportant plant hormone known to stimulate senescence, other hormoneslike jasmonate may also contribute to this process.

From the moment of harvest of the lettuce crop until the moment ofconsumption, the produce can be exposed to the different exogenousfactors contributing to senescence. These can be wounding duringharvesting and processing, darkness and nutrient deficiency duringstorage and ethylene during processing and storage. These factorsstrongly stimulate the post-harvest disorders which can become apparentas Russet Spotting and Yellowing. Although these effects are largelycosmetic the product becomes much less attractive and therebyunmarketable.

In order to counter the deterioration effects, many post-harvestmeasures can be taken which reduce these effects. For example, one canstore the harvested lettuce at low temperatures to retard senescence.Although this may reduce the rate of Yellowing, Russet Spotting may beenhanced. In addition, logistic measures may be implemented that reducethe transportation time required from the field to the consumer or thatprevent the lettuce from being stored in the vicinity of an ethylenesource. Furthermore, chemical treatments may be applied, which preventthe post-harvest deterioration, although food safety and consumeracceptance obviously become an issue.

Many of the post-harvest measures are successful to some extent butthere is certainly room for improvement. Moreover, costs involved may besubstantial, which is another reason to explore alternatives that reducethe need to apply post-harvest treatments. Preferably, a geneticsolution is found which reduces or eliminates the need to take theexpensive, preventive measures that are currently used to maintain thepost-harvest quality at a high level.

It is the object of the present invention to provide a screening methodto identify ethylene-insensitive plants. It is a further object of theinvention to provide plants that are obtainable by the method.

SUMMARY OF THE INVENTION

This object is achieved according to the invention by a method forscreening a population of plants for the presence therein of individualsthat show a reduced susceptibility to ethylene and physiologicaldisorders, in particular Russet Spotting and Yellowing, as compared to acontrol plant, which method comprises:

-   -   a) providing a population of seeds;    -   b) germinating the seeds in darkness and in the presence of        ethylene to obtain seedlings;    -   c) selecting seedlings that show longer hypocotyls than the        hypocotyls of an ethylene-sensitive control;    -   d) selfing the selected seedlings to produce seeds;    -   e) germinating one part of the seeds produced from each selected        seedling in darkness and in the presence of ethylene and another        part of the seeds from each selected seedling in darkness in        air; and    -   f) measuring the relative growth of the hypocotyls of the        seedlings germinated under ethylene versus the growth of the        hypocotyls of the seedlings germinated in air for distinguishing        plants that have a longer hypocotyl as compared to the original        ethylene-sensitive control both in ethylene and in air from        plants that have a longer hypocotyl as compared to the original        ethylene-sensitive control only under ethylene,        wherein plants that have a longer hypocotyl as compared to the        original ethylene-sensitive control only under ethylene are        identified as plants showing a reduced susceptibility to        ethylene and physiological disorders, in particular Russet        Spotting or Yellowing.

The invention provides a method of identifying a plant having reducedsusceptibility to a physiological disorder associated with ethylenesensitivity as compared to an ethylene-sensitive control plant, whichmethod comprises:

-   -   a) germinating a population of seeds in darkness and in the        presence of ethylene to produce seedlings;    -   b) selecting a seedling having elongated hypocotyls or roots as        compared to a seedling of the control plant;    -   c) self-fertilizing the selected seedling to produce seeds;    -   d) germinating a portion of the seeds in darkness and in the        presence of ethylene to produce Si seedlings and germinating a        portion of the seeds in darkness and in air to produce S2        seedlings; and    -   e) comparing hypocotyls of S1 seedlings to hypocotyls of S2        seedlings and to hypocotyls of control plant seedlings;        wherein if the hypocotyls of S1 seedlings are elongated as        compared to hypocotyls of S2 seedlings and control plant        seedlings, a plant having reduced susceptibility to a        physiological disorder associated with ethylene sensitivity as        compared to a control plant is identified.

The invention is based on the hypothesis that ethylene-insensitiveplants, in particular lettuce, would be resistant to post-harvestphysiological disorders such as Russet Spotting and Yellowing.

In a preferred embodiment, the method of the invention comprises thefurther step of testing plants that have been identified in the previoussteps as showing a reduced susceptibility to ethylene for theirresistance to Russet spotting and/or Yellowing.

Hypocotyl length and root length can be observed by comparison to thestandard and scored from 1, which means equal to the ethylene-sensitivestandard variety under ethylene, to 3, which means equal to theethylene-sensitive standard variety under air. If desired, measurementsin millimetres can be made. Using one of these measurements, simplestatistical analyses like a t-test, well-known to the person skilled inthe art, can be performed to establish whether a plant or group ofplants is significantly less sensitive to ethylene than theethylene-sensitive standard, like cv. ‘Troubadour’ (Rijk Zwaan, De Lier,NL). The applied significance level of a one-sided test is 0.001.

For non-naturally occurring plants, the statistical comparison ispreferably made to the hypocotyl lengths and optionally the root lengthsof the original variety (i.e., the starting material) from which thenon-naturally occurring plant was derived, which is the best availablestandard.

An ethylene-sensitive plant is a plant that shows a relatively strongresponse to ethylene. The relative response of a plant to ethylene canbe determined by comparing phenotypic characteristics of a plant grownunder ethylene versus air. Such phenotypic characteristics can bemanifested in many ways including but not limited to shoot and rootgrowth and development (so called triple response), yellowing, organabscission, fruit ripening and relative transcript abundance of ethyleneresponsive genes. The strength of the relative response of a plant toethylene depends on the specific conditions used to evoke such response,as well as on the genetic composition of the plant.

For finding ethylene-insensitive plants in existing plant material, oneor more representative samples of varieties, breeding lines and/or genebank accessions are selected. The statistical comparison is thensuitably made between the hypocotyl and root lengths of the individualaccession under investigation and the rest of the population. Whenstatistically testing individuals for significantly longer hypocotylsand/or roots multiple comparison tests may be needed to maintain properoverall significance levels, for example Dunnett's multiple comparisontest with one standard (Dunnett, C W, J. Amer. Statist. Assoc.50:1096-1121 (1955)).

The plants to be screened are usually leafy vegetable plants, inparticular plants belonging to the genus Lactuca and in particular tothe species Lactuca sativa.

The plant population is preferably a population of non-naturallyoccurring plants because the chances are higher to find a plant of theinvention in such variant population. However, any population of plantsmay be screened according to the invention.

The population of non-naturally occurring plants can comprise mutantplants, preferably obtained by a mutagenesis treatment using chemicalsand/or irradiation. Mutagenesis treatments are well-known and will befurther described below.

The concentration of ethylene in the initial step is at least 10μg/litre, preferably between 11 and 25 μg/litre. The concentration ofethylene in step d) is about 4 to 5 μg/litre.

The selection step in the screening method of the invention is based onthe elongation of the hypocotyls. Exposure of dark-grown germinatingseedlings to ethylene causes radial swelling of the hypocotyl andinhibition of root and hypocotyl growth. This phenomenon is generallyreferred to as the triple response. (See, for example, Guzman, P. &Ecker, J.; Plant Cell 2:513-523 (1990).) The reproducibility of thisresponse allows screening for plant that show an altered triple responsein the presence or absence of ethylene. In addition to or instead ofmeasuring the elongation of the hypocotyls, the selection according tothe invention may be based on one or more of the other elements of thetriple response test.

According to a further aspect, the invention provides plants showing areduced susceptibility to ethylene and physiological disorders, inparticular Russet Spotting and Yellowing, as compared to a controlplant, which plants are obtainable by subjecting a population of plantseeds to a screening method of the invention and selecting plants fromthe population that show longer hypocotyls in comparison with anethylene-sensitive control as plants showing a reduced susceptibility toethylene and physiological disorders, in particular Russet Spotting orYellowing.

The plant of the invention is preferably a leafy vegetable plant, inparticular a plant which belongs to the genus Lactuca and in particularto the species Lactuca sativa.

The invention further relates to plants that show a reducedsusceptibility to ethylene and physiological disorders, in particularRusset Spotting or Yellowing and are obtainable by crossing a plant ofthe invention with another plant of the same species. The feature“reduced susceptibility to ethylene and physiological disorders, inparticular Russet Spotting or Yellowing” can thus be brought into otherplants that originally do not have the feature. Whether the plantsresulting from such a cross are indeed plants of the invention can betested by subjecting these plants to the screening method of theinvention.

The invention further relates to progeny of a parent plant of theinvention that shows reduced susceptibility to ethylene andphysiological disorders, in particular Russet Spotting or Yellowing.Such progeny may be many generations removed from the parent. Providedthat the feature “reduced susceptibility to ethylene and physiologicaldisorders, in particular Russet Spotting or Yellowing” is present, theprogeny plant is a plant of the invention.

The invention further relates to parts of the plants of the invention.The plant parts are for example lettuce heads or leaves, such as babyleaves, processed heads or cut leaves.

Plant parts of the invention can be used in tissue culture to regenerateplants that have reduced susceptibility to ethylene and physiologicaldisorders, in particular Russet Spotting or Yellowing as found in theplant from which the tissue for the tissue culture is derived. Suchregenerated plants are also part of this invention.

The invention further relates to seed of a plant of the invention. Fromthe seed plants can be grown that also have the feature “reducedsusceptibility to ethylene and physiological disorders, in particularRusset Spotting or Yellowing.” Whether or not the seeds and thus theplants grown therefrom have retained that feature can be tested in thescreening method of the invention. The invention also relates to furthergeneration seeds that retain the reduced susceptibility to ethylene andphysiological disorders, in particular Russet Spotting or Yellowing asfound in the original seeds.

According to a further aspect thereof the invention relates to avegetable product, comprising a plant or part thereof of the invention.Preferably, the vegetable is a leafy vegetable, more in particular thevegetable is lettuce.

When screened in the method of the invention, the vegetable productshows reduced susceptibility to ethylene and physiological disorders, inparticular Russet Spotting or Yellowing.

It should be noted that the screening method of the invention is arelatively simple method. Plants of the invention that have a reducedsusceptibility to ethylene and physiological disorders, in particularRusset Spotting or Yellowing can be identified in any variant populationthat is sufficiently large. It does not require undue experimentation toreproduce the invention and the plants of the invention must thereforenot be construed as to be limited to the ones that are deposited.

As a result of their ethylene insensitivity, the lettuce plant materialof the invention and seed derived therefrom show a strong increase withrespect to their post-harvest quality especially related to thesenescence associated phenomena such as Russet Spotting and Yellowing.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

BRIEF DESCRIPTION OF THE DRAWINGS

In the examples reference is made to the following figure.

FIG. 1: Phenotype of mature heads of lettuce after exposure to ethyleneat 8° C. in the dark during 9 days. FIG. 1A shows a representativesample of ethylene-resistant (Ethylene R) and ethylene-sensitive control(-control) heads of the variety Troubadour. FIG. 1B shows a similarpicture of the mutants derived from the variety Apache.

DETAILED DESCRIPTION OF THE INVENTION

As ethylene is the most important plant hormone in stimulatingsenescence and as Russet Spotting and Yellowing are associated withsenescence, a genetic approach was taken in which genetic variants wereproduced and selected for ethylene insensitivity. It was found that thisindirectly leads to the identification of plants that are affected intheir post-harvest senescence response.

Detailed studies using plant model species such as Arabidopsis thalianaas well as crop species have provided information on the biochemicalpathways involved in the biosynthesis and perception of ethylene. Manyallelic forms of genes have been characterised for their role in thisrespect providing an intricate picture of ethylene function in plants.Ethylene, like other plant hormones, plays an important role in manyphysiological processes embedded in complicated interactive regulatorynetworks.

The spatial and temporal activity of the hormone is inter aliadetermined at the level of gene expression underlying ethylenebiosynthesis, hormone perception, signal transduction and activation ofdownstream effector proteins.

Allelic variants of genes involved at different levels can determine thestrength of the response as well as the level of cross-talk to othersignaling pathways. As ethylene biosynthesis and perception are poorlycharacterised in lettuce and in order to allow the identification ofdifferent response strengths and their underlying allelic variants itwas reasoned that an unbiased approach may be more successful in thisrespect as compared to a targeted gene modification approach.

Such unbiased approach encompasses preferably a chemical or physicalrandom mutagenesis procedure combined with an efficient phenotypicscreening and selection procedure based on a response of etiolatedseedlings to ethylene. Such seedling-based selection system is by farmore efficient in terms of numbers of plants which can be assessed perman hour as compared to the use of mature lettuce heads. Furthermore,the time to produce plant material for screening is obviously much morereduced in the case of seedlings as compared to mature heads.

A further advantage of this approach is the use of the selectionconditions at the seedling stage as predictive phenotypic marker for thepost-harvest trait in consecutive generations once a successful eventhas been identified. A clear risk exists in the fact that selection atthe seedling stage may not lead to genetic variants which express theselected trait at the mature, post-harvest level. Furthermore it isrecognised that plant hormones like ethylene are involved in manyphysiological processes which may lead to pleitotropic effects. Thesecan either be positive or negative depending on the crop and itscultivation conditions. The approach taken therefore preferablycomprises the following steps:

-   -   1. Generation of a variant population of plants.    -   2. The set-up of an efficient phenotypic screen in which        selection is based on a response of etiolated seedlings of        plants, in particular lettuce, to ethylene which is        characterised by a strong reduction of the hypocotyl elongation,        a shortened, thickened root and an exaggerated apical hook        curvature. This response is called the triple response which is        typical for etiolated seedlings and found to occur to various        degrees in many plant species when exposed to atmospheres        containing ethylene. (See, for example, Ecker J., Science        268(5211):667-675 (1995).)    -   3. Characterisation of the plants modified in at least one test        of their triple response with respect to post-harvest        deterioration which comprise Russet Spotting and Yellowing.        Optionally, determination of pleiotropic effects of the        modification to exclude negative pleitropic effects that would        affect the value of the plants.

“Variant plants” or “non-naturally occurring plants” are plants producedby human intervention. Examples of such plants are mutant plants,genetically modified plants produced, for instance, using recombinantmethods, and plants resulting from intentional cross-breeding orselling.

In the screening method of the invention the population of plant seedsto be tested is preferably a population of mutant seeds that can beobtained by a method comprising:

-   -   a) treating M0 seeds of a plant species to be modified with a        mutagenic agent to obtain M1 seeds;    -   b) growing plants from the thus obtained M1 seeds to obtain M1        plants;    -   c) producing M2 seeds by self-fertilisation of M1 plant; and    -   d) optionally repeating step b) and c) n times to obtain M1+n        seeds.

The M1+n seeds thus obtained are then germinated in darkness and in thepresence of high ethylene concentration to obtain seedlings.Subsequently, seedlings are selected that do not show a response toethylene. The response to be measured is the development of hypocotylsthat are elongated as compared to the hypocotyls of anethylene-sensitive seedling.

Then, progeny of each selected plant is split up and grown in the dark:half of the progeny under ethylene, half of the progeny under air.Relative growth of the hypocotyl under ethylene versus air is measuredfor each progeny under both conditions. These observations are used todistinguish progenies which have a longer hypocotyl than the originalethylene-sensitive control under ethylene, but also under air, and whichcan therefore be concluded to be ethylene-sensitive and not desired,from the progenies truly have a reduced susceptibility to ethylene. Anexample of a well-known mutagen is ems. Ems alkylates primarily Gresidues of a DNA strand which during DNA replication causes pairingwith T instead of C. Therefore, GC basepairs change to AT basepairs at afrequency which is determined by the effective dose of ems and theactivity of the mismatch repair system of the plant. The effective doseof ems depends on the concentration used, the seed size and otherphysical properties and the time of incubation of the seeds in the emssolution. The seeds which have been treated with ems are typicallycalled M1 seeds. As a consequence of the treatment, the tissues of theM1 seeds contain random point mutations in the genomes of their cellsand those present in the subpopulation of cells which will form thegermline tissue (germinal cells) will be transferred to the nextgeneration which is called M2. Mutations or combinations thereof whichare haplo-insufficient thereby causing sterility or which induce embryolethality will not be transferred to the M2 generation.

A similar procedure as described above for the use of ems applies forother mutagenic agents as well. The M2 population can be used inscreening procedures aimed at a reduced triple response of etiolatedseedlings.

Other mutagenic agents, in particular alkylating mutagenic agents, arediethyl sulfate (des), ethyleneimine (ei), propane sultone,N-methyl-N-nitrosourethane (mnu), N-nitroso-N-methylurea (NMU),N-ethyl-N-nitrosourea (enu), sodium azide.

Alternatively, the mutations are induced by means of irradiation, whichis for example selected from x-rays, fast neutrons, UV irradiation.

In another embodiment of the invention the non-naturally occurringplants are produced by means of genetic engineering, such as by means ofuse of chimeric oligonucleotides, homologous recombination, introductionof modified target genes which compete with the endogenous product,downregulation through RNA interference, etc.

The technology to modify gene targets residing in the genome of a plantin a specific manner is known to the person skilled in the art. Forexample, chimeric oligonucleotides have been demonstrated to beeffective mutagens with a specific mode of action. Another approach isto modify gene targets through homologous recombination or genetargeting. Using such approach, a fragment of a gene is exchanged by anintroduced DNA fragment containing a desired modification. Transgenicapproaches are also feasible in which modified target genes areintroduced which compete with the endogenous product. This may lead todominant negative effects. Moreover specific downregulation of theexpression of genes is feasible through RNA interference.

Where mutagenic oligonucleotides, gene targeting or transgenicapproaches are used to modify a genetic factor involved in ethylenefunction, obviously, the primary structure of the relevant genes shouldbe known. Currently however, for lettuce knowledge on such genes islimited.

Preferably, the invention further comprises pyramiding alleles ofreduced susceptibility towards ethylene.

Production of M1 and M1+n seeds is suitably effected by means ofself-pollination.

The screening method of the invention may also be used to identifynaturally occurring, wild type plants with the desired phenotype ofreduced wound-induced discolouration. Once identified, these plants canbe crossed or selfed and the non-naturally occurring progeny selectedfor the desired phenotype.

The invention further relates to plants or plant parts, which have intheir genome genetic information which is responsible for the reducedsusceptibility towards physiological disorders such as Russet Spottingand Yellowing and is found in the genome of a lettuce plant as listed inTable 1.

Progeny of the plants as claimed are also part of this invention.“Progeny” as used herein is intended to encompass all plants having thesame or a similar reduced susceptibility towards ethylene andphysiological disorders, in particular Russet Spotting or Yellowing, asthe original plants described herein and being derived therefrom in anyway, such as by sexual reproduction, such as self-fertilisation orcross-fertilisation with another plant of the same genus, or vegetativereproduction, such as cutting, tissue culture, haploid culture,protoplast culture, protoplast fusion or other techniques. Such progenyis thus the first generation of plants as identified according to theinvention, as well as the first generation of plants derived by one ormore of these techniques. Also included in the invention is everyfurther generation of plants derived by one or more of these techniques,provided that the derived plants have reduced susceptibility.

To determine the response of the etiolated lettuce seedlings towardsethylene, use was made of specially designed plastic containers in whichlettuce seedlings were grown on filter papers under an atmosphere inwhich ethylene levels can be varied. It was indeed found that thelettuce seedlings responded to the presence of ethylene by a reducedelongation of the hypocotyl, which in principle would allow selectionfor ethylene-insensitive variants in case such variants reside in theavailable population and in case the insensitivity is expressedphenotypically at the seedling level under the experimental conditionswhich were applied.

By growing large numbers of etiolated lettuce seedlings from apopulation containing randomly induced mutations under an ethylenecontaining atmosphere, it was found that seedlings showing reducedethylene sensitivity as compared to the ethylene sensitivity of thestarting population can be obtained and selected. Seedlings wereselected showing hypocotyl elongation comparable to a situation in whichthe atmosphere was composed of air without ethylene. The seedlingsidentified in this manner were characterized as being insensitive toethylene.

To confirm that the ethylene-insensitive variants are resistant toRusset spotting and Yellowing the variants identified in the screen aretested for their resistance to Russet spotting and/or Yellowing.

A Russet spot test suitably comprises storing harvested mature heads ina closed container at a temperature of 8° C. in the dark, and exposingthem to ethylene gas at a concentration of between 6 and 7 vpm (volumeparts per million) and assessing the presence of symptoms of Russet spotafter 7 days, preferably after 9 days. Suitably, an ethylene-sensitivecontrol head is incubated together with the plants to be tested andRusset spotting of the plants to be tested is compared therewith.

A Yellowing test comprises storing mature heads of the lettuce plants tobe tested in an ethylene-free storage chamber at 8° C. and assessing theyellowing of the base leaves after 10 days, preferably after 14 days.Yellowing resistant plants are plants that do not show yellowing of thebase leaves after 14 days. Suitably, an ethylene-sensitive control headis incubated together with the plants to be tested and the Yellowingresponse of the plants to be tested is compared therewith.

As illustrated in the Examples, it has been demonstrated for the firsttime that ethylene-insensitivity leads to resistance to Russet spottingand to resistance to Yellowing that is not induced by ethylene.

Surprisingly, seedlings which showed an elongation response which wasreduced as compared to the control conditions but which were longer ascompared to the sensitive controls were also found which were consideredto be insensitive to ethylene as well, albeit partially.

The variable levels of ethylene insensitivity that were observed mayreflect either the presence of different mutant loci or differentallelic forms of identical loci affecting this trait in the originalpopulation.

In case of recessive mutation, these two possibilities can easily bedistinguished by carrying out allelism assays which comprises thecrossing of the two mutant events and determining the phenotype of thehybrid. In case of allelism of the mutations, the ethylene insensitivitywill be apparent in the F1 whereas in case the phenotype in the mutantsis determined by different recessive loci this will not be the case.

As random mutagenesis was applied as a preferred means to generate thestarting population, mutations in the genetic background may alsocontribute to the variation of the seedling phenotype under theexperimental conditions. In order to discriminate between singlemutations of different strength and a combined effect of mutation in thegenetic background, backcrosses should be performed to create uniformgenetic backgrounds for the different ethylene-insensitive events. Suchprocedure is further relevant in order to determine whether mutations atspecific loci involved in ethylene sensitivity display pleiotropiceffects.

The M2 plants thus selected on the basis of a reduced response toethylene were used to grow M3 seeds. In some cases M3-plants grown fromthe M3-seeds were selected for reduced ethylene sensitivity in a tripleresponse test and selfed to produce M4-seeds. This was even repeateduntil M5 in a few cases.

In one case, an M3-plant grown from M3-seed was selected for reducedethylene sensitivity in a triple response test, and crossed with anethylene-sensitive and Russet spot sensitive parent to obtain F1-seed.An F1-plant grown from the F1-seed was selfed to produce F2-seeds. AnF2-plant grown from these F2-seeds was selected for reduced ethylenesensitivity in a triple response test, and selfed to produce F3-seeds.This F3-line was added to the set of M3 and M4 inbred lines.Subsequently, the inbred lines descending from the ethylene-insensitiveevents were re-evaluated for their response to ethylene. The level ofinsensitivity of each inbred line was scored on the basis of therelative growth of the seedling under an ethylene containing atmosphereversus air. Based on this criterion 12/54 lines that were scoredethylene-insensitive earlier were now classified as being sensitive.

These false positives during the initial screen at the M2 level, canreadily be eliminated during the re-evaluation of the events in the nextgeneration.

The inbred lines that showed a confirmed and significant ethyleneinsensitivity in the ethylene test were resown and grown in a greenhouseunder regular lettuce production conditions to produce mature heads thatwere assessed for Russet Spotting and Yellowing. (See also theExamples.)

As negative controls, ethylene-sensitive plants that originate from thepopulation which was used to select the ethylene-insensitive events weregrown. Surprisingly, the seeds of all ethylene-insensitive mutantsgerminated normally i.e. comparable to the seeds of a near-isogenicethylene-sensitive control plant when planted in potting soil.

This contrasts sharply to the situation in other plant species, such asArabidopsis thaliana, which show a strong reduction in germinationcapacity when planted in potting soil (Harpham, N. J. V. et al. (1991)Annals of Botany 68, 55-61). Apparently for lettuce it is possible togrow ethylene-insensitive mutants according to a normal cultivationpractice, which, in many cases, includes sowing in potting soil blocksor potting soil plugs.

After cultivation, mature heads were harvested and exposed to ethylene.One week of post-harvest incubation of heads at 8° C. under an ethylenecontaining atmosphere resulted in a strong induction of Russet Spottingof the heads of the ethylene-sensitive control plants. However, 29 outof 37 ethylene-insensitive events which were assessed showed no sign ofRusset Spotting at all which surprisingly demonstrates that ethyleneresistance which was selected for at the seedling level can reducephysiological disorders at the mature plant level, even at thepost-harvest stage.

Yellowing resistance could be demonstrated for a number ofethylene-insensitive events under ethylene-free storage conditions atsub-optimal temperature. This is surprising, because until now Yellowingresistance has only been reported in the presence of ethylene. (Saltveitet al., Postharvest Biology and Technology 27:277-283 (2003).)

With the screening method of the invention, ethylene-insensitive mutantswere identified and selected. Seeds of these mutants were deposited withNCIMB Ltd., Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen,AB21 9YA UK on 3 Jan. 2007 and has been given the accession numberslisted in Table 1. Details about seed descendance of the deposits aregiven in Example 3 and in Example 4. These deposits are made becausethey have the single specific characteristic of significantly reducedsusceptibility to ethylene. They were not tested for DUS-criteria forvariety registration, i.e. distinguishability, uniformity, stability onall registration characteristics, and are not expected to meet thesecriteria in any way.

TABLE 1 Internal ref. NCIMB accession Plant no. (=NCIMB ref.) number00D.7856 07D.826509 41449 00D.6876 07D.826514 41450 00D.7871 07D.82650241448 00D.6883 07D.826522 41451 00D.6896 07D.826540 41452 00D.784507D.826542 41453

The present invention will be illustrated in the Examples that followand that are not intended to limit the invention in any way. More inparticular, the experiments in the Examples are performed with lettucebut the invention is more broadly applicable to other plant species thatencounter similar post-harvest difficulties when contacted withethylene.

EXAMPLES Example 1 Genetic Modification of Lettuce by Ethyl MethaneSulfonate (ems)

Seeds of the lettuce varieties Troubadour, Apache and Yorvik (all fromRijk Zwaan, De Lier, The Netherlands) were treated with ems bysubmergence of approximately 2000 seeds per variety into an aeratedsolution of either 0.05% (w/v) or 0.07% (w/v) ems during 24 hours atroom temperature.

Approximately 1500 treated seeds per variety per ems dose weregerminated and the resulting plants were grown in a greenhouse in theNetherlands from May to September to produce seeds.

After maturation, M2 seeds were harvested and bulked in one pool pervariety per treatment. The resulting 6 pools of M2 seeds were used asstarting material to identify the individual M2 plants containingreduced susceptibility alleles.

The efficacy of the genetic modification procedure was assessed bydetermining the occurrence of bleached plants, which is indicative forchlorophyll loss due to modifications in genes directly or indirectlyinvolved in the formation or accumulation of chlorophyll. In all 6 poolsof M2 seeds individual plants, which are bleached, were observed whichdemonstrates that the applied treatments result in geneticmodifications.

Example 2 Identification of Lettuce Plants which have Obtained ReducedSusceptibility Alleles for Ethylene

M2 lettuce seeds were germinated on paper in a small plastic containerwith an ethylene concentration of 10 20 vpm (volume parts per million)at 16° C. in dark. 1 vpm contains 0.41 μmol/litre or 1.14 μg/litre.Ethylene-insensitive mutants were compared to ethylene-sensitivecontrols, and selected on the basis of elongated hypocotyl and/or root(i.e. triple response test). These ethylene-insensitive mutants weregrown to produce M3 lines by self fertilisation. These M3 lines werere-tested with the triple response test to confirm ethyleneinsensitivity.

When a line was segregating for ethylene insensitivity, plants wereselected followed by one or two additional cycles of inbreeding, and afinal triple response test to select homozygous ethylene-insensitivelines, if possible.

In one case, an M3-plant grown from M3-seed was selected for reducedethylene sensitivity in a triple response test, and crossed with anethylene-sensitive and russet spot sensitive parent Troubadour to obtainF1-seed. An F1-plant grown from the F1-seed was selfed to produceF2-seeds. An F2-plant grown from these F2-seeds was selected for reducedethylene sensitivity in a triple response test, and selfed to produceF3-seeds. The resulting F3-line was added to the set of 53 M3, M4, andM5 inbred lines. In this case the F3-line was the only representative ofthe original M2-mutant plant, because there were no selfed seeds leftfrom this plant.

This set of 54 M3 and M4 lines from Example 1 were germinated on peatblocks in a closed container under ethylene concentration between 4 and4.5 vpm in the dark. Ethylene-insensitive mutants were identified bytheir longer hypocotyls in comparison with sensitive control varieties(Troubadour, Apache, Yorvik, Sensaï). Results are presented in Table 1.42 Out of 54 lines that were identified by the triple response testappeared to be at least partial ethylene-insensitive in the ethylenetest. These lines represent 40 M2 plants because two M2 plants wererepresented twice. Table 2 shows the results.

TABLE 2 Ethylene response of mutant lines in a closed container test anda triple response test (TRT). Observations on hypocotyl and root areindicated separately, when no consistent response is shown. Plot Seedno. Origin Ethylene TRT 1. Yorvik S S 2. Troubadour S S 3. Apache S S 4.00D.88531 Y S R/S hypo; R root 5. 00D.88539 Y S S 6. 01D.85717 Y S R 7.01D.85720 Y S R 8. 01D.85732 Y S S 9. 00D.88533 Y S S 10. 00D.88538 Y SR 11. 03D.74008 Y R S 12. 02D.90749 Y S S 13. 00D.88550 Y S S 14.01D.85739 Y S S 15. 00D.88565 T R R 16. 02D.91445 T R R 17. 00D.88577 TR/S R hypo; S root 18. 02D.91446 T R R 19: 02D.91447 T R 20. 01D.85754 TS? R/S 21. 02D.90047 T R R 22. 01D.85758 T R R 23. 00D.88564 T S S 24.02D.91442 T R R 25. 00D.88569 T R R 26. 00D.88573 T R R 27. 00D.88578 TR R 28. 00D.88582 T R R 29. 01D.85748 T R/S R/S 30. 01D.85750 T S R 32.01D.85755 T R/S R/S 33. 01D.85762 A Partial R R hypo; S root 34.03D.90452 A R R 35. 01D.85764 A Partial R hypo; R/S S root 36. 02D.90070A R R 37. 03D.90457 A R R 38. 01D.85768 A R R 39. 02D.90128 A R R 40.02D.90129 A R R 41. 02D.90130 A R R 42. 03D.90462 A R R 43. 03D.90464 AR R 44. 02D.90133 A R R 45. 02D.90134 A R R 46. 00D.88600 A R/S R 47.  02D.91479+ A R R 48. 01D.85772 A R S hypo; R root 49.  04D.800957 A RS hypo; R root 50. 02D.90091 A R R 51.  04D.800960 A R S hypo; R root52.  04D.800963 A R R 53. 01D.85780 A R R 54. 01D.85756 T R R 55.02D.90036 T Partial R S hypo; R root 56.  04D.801660 Y Partial R R/Shypo; S root 58.  04D.800900 T R S hypo; R root 59. 03D.90323 T R R 60.Sensai S S Intermediate response is indicated as partial. R =ethylene-insensitive; S = ethylene-sensitive; R/S = segregating; T =Troubadour; A = Apache; Y = Yorvik; hypo = hypocotyl

Example 3 Identification of Lettuce Plants which have Obtained ReducedSusceptibility Alleles for Russet Spot

Thirty-seven ethylene-insensitive lines from the forty-two lines foundin Example 2 were sown in a greenhouse to produce mature heads underregular lettuce production conditions (location: Maasdijk, theNetherlands; sowing on day 1, transplanting on day 39, harvesting on day99). The harvested mature heads were stored in a closed container at atemperature of 8° C. in the dark. They were exposed to ethylene gas at aconcentration of between 6 and 7 vpm (volume parts per million). One vpmcontains 0.41 μmol/liter or 1.14 μg/liter. After 9 days, plantsdisplaying Russet Spotting were identified. All control plants, exceptYorvik displayed Russet Spotting. 29 Out of 37 lines tested showedabsence of Russet Spotting or less Russet Spotting as compared to theoriginal variety (Table 3).

Six lines were chosen for multiplication and deposit at NCIMB Ltd,Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen: AB21 9YA, UK.The first line is numbered 02D.91445. It is an M4-line descending fromthe ethylene-insensitive Troubadour-M2-plant 00D.7856. Anethylene-insensitive M4-plant was selected from 02D.91445 in the closedcontainer test in Example 2 and selfed to produce M5-seeds. SixteenM5-plants were grown from these seeds to produce an M6 seed lot byselfing. This seed lot is numbered 07D.826509 and is deposited asNCIMB-number 41449 (first line).

The second line is numbered 02D.90047. It is an M4-line descending fromthe ethylene-insensitive Troubadour-M2-plant 00D.6876. Anethylene-insensitive M4-plant was selected from 02D.90047 in the closedcontainer test in Example 2 and selfed to produce M5-seeds. SixteenMS-plants were grown from these seeds to produce an M6 seed lot byselling. This seed lot is numbered 07D.826514 and is deposited asNCIMB-number 41450 (second line). The absence of russet spotting forthis origin was established by testing the M5-line 03D.90323, descendingfrom an M4-plant of 02D.90047.

The third line is numbered 00D.88578. It is an M3-line descending fromthe ethylene-insensitive Troubadour-M2-plant 00D.7871. Sixteen M3-plantswere grown from the 00D.88578 to produce an M4 seed lot by selfing. Thisseed lot is numbered 07D.826502 and is deposited as NCIMB-number 41448(third line).

The fourth line is numbered 03D.90452. It is an M5-line descending fromthe ethylene-insensitive Apache-M2-plant 00D.6883. Anethylene-insensitive M5-plant was selected from 03D.90542 in the closedcontainer test in Example 2 and selfed to produce M6-seeds. SixteenM6-plants were grown from these seeds to produce an M7 seed lot byselfing. This seed lot is numbered 07D.826522 and is deposited asNCIMB-number 41451 (fourth line).

The fifth line is numbered 01D.85780. It is an M3-line descending fromthe ethylene-insensitive Apache-M2-plant 00D.6896. Anethylene-insensitive M3-plant was selected from 01D:85780 in the closedcontainer test in Example 2 and selfed to produce M4-seeds. SixteenM4-plants were grown from these seeds to produce an M5 seed lot byselfing. This seed lot is numbered 07D.826540 and is deposited asNCIMB-number 41452 (fifth line).

The sixth line is numbered 04D.801660. It is an F3-line descending froma cross between the ethylene-insensitive Yorvik-M3-plant 02D.8484 and aplant of the ethylene-sensitive variety Troubadour. The M3-plant02D.8484 descended from the ethylene-insensitive Yorvik-M2-plant00D.7845. An ethylene-insensitive F3-plant was selected from 04D.801660in the closed container test in Example 2 and selfed to produceF4-seeds. Sixteen F4-plants were grown from these seeds to produce an F5seed lot by selfing. This seed lot is numbered 07D.826542 and isdeposited as NCIMB-number 41453 (sixth line).

FIG. 1 shows resistant and control plants.

Example 4 Identification of Lettuce Plants that Show Less Post-HarvestYellowing

Ethylene-insensitive lines grown under greenhouse conditions asdescribed for Example 3 were harvested and mature trimmed heads werestored in a ethylene free storage chamber at 8° C. After two weeks baseleaves of the green control varieties Yorvik and Troubadour started toturn yellow. At that moment and even 1 week later three lines appearedto have less yellow base leaves than their origin varieties, which werethe controls used in this trial. These lines were numbered 04D.800900,03D.90323, 04D.801660. Although a relationship is reported between leafyellowing and ethylene presence (Saltveit et al. (2003) PostharvestBiology and Technology 27:277 283), it is surprising that even inconditions without ethylene, some of the ethylene-insensitive lines areexpressing a less yellowing phenotype.

TABLE 3 Observations of russet spotting on mature heads after 9 days ofstorage in ethylene conditions. Russet Seed no. Ethylene TRT Spot01D.85762 Partial R R hypo; 2 S root 03D.90452 R R 0 01D.85764 R/S Rhypo; 1 S root 02D.90070 Partial R R 0 03D.90457 R R 0 01D.85768 R R 002D.90128 R R 2 02D.90129 R R 2 02D.90130 R R 2 03D.90462 R R 2 Apache SS 2 03D.90464 R R 0 02D.90133 R R 2 02D.90134 R R 2 00D.88600 R/S R NA02D.91479 R R 0 01D.85772 R S hypo; 0 R root  04D.800957 R S hypo; 0 Rroot 02D.90091 R R 0  04D.800960 R S hypo; 0 R root  04D.800963 R R 201D.85780 R R 0 Apache S S 2 00D.88565 R R 0 02D.91445 R R 0.5 00D.88577R/S R hypo; 0 S root 02D.91446 R R 0 02D.91447 R R 0 01D.85754 S R/S 001D.85756 R R 0 02D.90047 R R NA 01D.85758 R R 0 02D.91442 R R 0Troubadour S S 1 00D.88569 R R 0 00D.88573 R R 0 00D.88578 R R 000D.88582 R R 0 01D.85748 R/S R/S 0/1 02D.90036 Partial R S hypo; NA Rroot 01D.85755 R/S R/S NA  04D.800900 R S hypo; 0.5 R root 03D.90323 R R0  04D.801660 Partial R R/S hypo; 0/2 S root Yorvik S S 0 0 = symptomsabsent; 1 = weak symptoms; 2 = strong symptoms; NA = not available

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theappended claims is not to be limited to particular details set forth inthe above description, as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.Modifications and variations of the method and apparatuses describedherein will be obvious to those skilled in the art, and are intended tobe encompassed by the following claims.

1. A method of identifying a plant having reduced susceptibility to aphysiological disorder associated with ethylene sensitivity as comparedto an ethylene-sensitive control plant, which method comprises: a)germinating a population of seeds in darkness and in the presence ofethylene to produce seedlings; b) selecting a seedling having elongatedhypocotyls or roots as compared to a seedling of the control plant; c)self-fertilizing the selected seeding to produce seeds; d) germinating aportion of the seeds in darkness and in the presence of ethylene toproduce S1 seedlings and germinating a portion of the seeds in darknessand in air to produce S2 seedlings; and e) comparing hypocotyls of S1seedlings to hypocotyls of S2 seedlings and to hypocotyls of controlplant seedlings; wherein if the hypocotyls of S1 seedlings are elongatedas compared to hypocotyls of S2 seedlings and control plant seedlings, aplant having reduced susceptibility to a physiological disorderassociated with ethylene sensitivity as compared to a control plant isidentified.
 2. The method of claim 1, wherein the physiological disorderassociated with ethylene sensitivity is Russet Spotting or Yellowing. 3.The method of claim 1, wherein the plant is a leafy vegetable plant. 4.The method of claim 1, wherein the plant belongs to the genus Lactuca.5. The method of claim 4, wherein the plant is Lactuca sativa.
 6. Themethod of claim 1, wherein the plant is from a germplasm collection. 7.The method of claim 1, wherein the plant is a non-naturally occurringplant.
 8. The method of claim 7, wherein the non-naturally occurringplant is a mutant plant obtained by chemical mutagenesis and/orirradiation.
 9. The method of claim 7, wherein the plant is a transgenicplant.
 10. The method of claim 1, wherein the concentration of ethylenein step a) is at least 10 μg/litre.
 11. The method of claim 10, whereinthe concentration of ethylene is 11-25 μg/litre.
 12. The method of claim1, wherein the concentration of ethylene in step d) is about 4-5μg/litre.
 13. A non-naturally occurring plant, or part thereof,identified by the method of claim 1, wherein the plant has reducedsusceptibility to a physiological disorder associated with ethylenesensitivity as compared to an ethylene-sensitive control plant.
 14. Theplant of claim 13, wherein the plant is a leafy vegetable plant.
 15. Theplant of claim 13, wherein the plant belongs to the genus Lactuca. 16.The plant of claim 15, wherein the plant is Lactuca sativa.
 17. Theplant of claim 13, wherein the plant part is a leaf, a head, a cut leaf,a baby leaf or a processed head.
 18. A seed of the plant of claim 13.19. A seed deposited with NCIMB having an accession number selected fromthe group consisting of: i) accession number 41449, ii) accession number41450, iii) accession number 41448, iv) accession number 41451, v)accession number 41452, and vi) accession number 41453; or a seed havingall of the identifying characteristics thereof.
 20. A plant, or partthereof, produced from the seed of claim 19, having reducedsusceptibility to a physiological disorder associated with ethylenesensitivity as compared to an ethylene-sensitive control plant.
 21. Aplant, or part thereof, produced by propagation of and/or breeding withthe plant of claim 20, and having all the identifying characteristics ofthe plant of claim 20.